Method of modulating a testicular carnitine transporter

ABSTRACT

It is intended to identify and provide a novel carnitine transporter gene participating in carnitine transport in the testis and epididymis and carnitine transporter which is a protein encoded by the gene. A protein comprising the amino acid sequence represented by SEQ ID NO:2 or an amino acid sequence derived therefrom by deletion, substitution or addition of one to several amino acids and being capable of transporting carnitine or its analog; and a gene encoding this protein.

TECHNICAL FIELD

The present invention relates to a gene participating in sodiumdependent transport of carnitine and its analog in the testis and theepididymis and a polypeptide encoded by the gene.

BACKGROUND ART

Carnitine has an important function in beta-oxidation of fatty acids inevery organ of a living body. Cells in every organ produce ATP(adenosine triphosphate), which is the energy source for the cells, inmitochondria owing to the beta-oxidation of fatty acids. An acyl groupof the fatty acids is transported in the mitochondria through acarnitine action to be transformed into acyl Co-A, and then the acylCo-A is beta-oxidized to generate ATP. Many carnitines are diet-induced,and, though a part thereof is biosynthesized in the liver and the brain,almost all the cells need to uptake carnitines from outside; therefore,it has been assumed that a carnitine-specific transporter exists in thecell membrane according to past physiological studies.

Studies have been conducted on the carnitine uptake by various cells bythe use of experiment systems employing the removed organ perfusionmethod or the isolated cell membrane vesicular system. However, it hasbeen difficult to analyze the system of carnitine transport through cellmembrane in detail by the conventional methods, and there has been ademand for isolation and analysis of the transporter itself.

The rat CT1 (Sekine, T. et al., Biochem. Biophis. Res. Commun., Vol.251, pp. 586-591, 1998) has been isolated as the carnitine transporter.The hOCTN2 (Tamai, I. et al., J. Biol. Chem., Vol. 273, pp. 20378-20382,1998) has been reported to be a human homologous gene of the CT1. Thecarnitine transporters are widely distributed in organs of the wholebody to contribute to the carnitine uptake by various cells. They existalso in the kidney to carry out a function of reabsorption of carnitinefrom the renal tubules.

According to the past studies, it has been clarified that a carnitineconcentration in the testis and the epididymis is the highest in theliving body and reached the 2,000 times of the serum level depending onthe specie of the carnitine. Since the duct of epididymis is an organgoverning nutrition and maturity of sperms, the relationship betweencarnitine and sperm function has attracted attention.

It has been clarified that the sperms produced in the testis arepremature and low in motility and lacks in fertilizability at thatstage. The sperms leaving the testis move to the epididymis andsubjected to various modifications therein and matured. Further,flagellar movement is necessary for the sperm fertility, which requiresenormous energy. It is reasonable to consider that a large amount ofcarnitine is required for the production of ATP which is the energysource for the flagellar movement.

Actually, a study revealing a positive relationship between thecarnitine concentration in the human semen and the number and motilityof sperms has been reported. Also, in the clinical medicine, it has beenreported that carnitine administration had resulted in an increase infertilization probability of idiopathic infertility patients.

Thus, the carnitine transport specific to the testis and the epididymishas been considered to have an important function in the maturity andthe fertilizability of sperms and has been studied by the experimentsystems employing the removed organ perfusion method or the isolatedcell membrane vesicular system. Though the existence of an activecarnitine transport system has been clarified from the studies, itsmolecular substance has been indefinite.

Organic anion transporters OAT1 (organic anion transporter 1) (Sekine,T. et al., J. Biol. Chem., Vol. 272, pp. 18526-18529, 1997), OAT2(Sekine, T., FEBS letter, Vol. 429, pp. 179-182, 1998), OAT3 (Kusuhara,H. et al., J. Biol. Chem., Vol. 274, pp. 13675-13680, 1999), and OAT4(Cha, S. H. et al., J. Biol. Chem., Vol. 275, pp. 4507-4512, 2000),which have the central role in drug transport in the kidney, liver,brain, and placenta, have been isolated and reported by some of theinventors of the present invention. These transporters belonging to anOAT family are transporters that can transport many different organicanions in chemical structure and also transport of various anionicdrugs.

Recently, an OCT (organic cation transporter) family which recognizesorganic cations also has recently been identified. Since carnitine is anorganic ion having both of a positive charge and a negative charge, itis assumed that the carnitine transporter is positioned between the OATfamily and the OCT family from the point of molecular evolution.

In view of these facts, the present inventors have presumed that thecarnitine transporter specific to the testis and the epididymis belongsto the organic ion transporter family.

DISCLOSURE OF THE INVENTION

An object of the present invention is to identify and provide a novelcarnitine transporter gene participating in carnitine transport in thetestis and the epididymis and a carnitine transporter which is apolypeptide encoded by the gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A) a result and (B) a time dependence of an experiment oncarnitine uptake by oocytes into which a cRNA of human CT2 gene wasinjected.

FIG. 2 shows (A) a result of a Michaelis-Menten kinetics experiment oncarnitine uptake by oocytes into which the cRNA of human CT2 gene wasinjected and (B) a result of D-carnitine inhibition effect inL-carnitine transport by the CT2.

FIG. 3 shows (A) a result of influences of added salts and (B) a resultof influences of pH of an experiment on carnitine uptake by oocytes intowhich the cRNA of human CT2 gene was injected.

FIG. 4 shows a result of influence of additions of carnitine analogs toa system of an experiment on carnitine uptake by oocytes into which thecRNA of human CT2 gene was injected.

FIG. 5 is a photo substituted for drawing, showing a result of analysesof CT2 gene mRNA expressions in the human organs by the northernblotting method. FIGS. 6A-6F show the results of immunohistologicalanalyses of CT2 protein expression in the testis and the epididymis, anexperiment described in Example 1 (8): FIGS. 6A-6C are photos of testisand FIGS. 6D-6E are photos of epididymis; FIGS. 6A and 6D show testisand epididymis, respectively, which were incubated with polyclonalanti-CT2 antibody and then reacted with anti-rabbit lgG antibody labeledwith peroxidase and stained by diaminobenzidine; FIGS. 6B and 6E areamplified pictures of FIGS. 6A and 6D, respectively. FIGS. 6C and 6Fshow testis and epididymis, respectively, which were incubated withpolyclonal anti-CT2 antibody and then reacted with anti-rabbit lgGantibody labeled with peroxidase and stained by diaminobenzidine.

FIG. 7 shows a result of analyzing a structure of a human genome of CT2gene.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to (1) a protein having an ability totransport carnitine or its analog, comprising an amino acid sequencerepresented by SEQ ID NO: 2 or an amino acid sequence derived from thesaid amino acid sequence by deletion, substitution, or addition of oneto several amino acids.

Also, the present invention relates to (2) a gene encoding theabove-described protein.

Further, the invention relates to (3) a gene comprising a DNA having abase sequence represented by SEQ ID NO. 1 or a DNA encoding a proteinwhich hybridizes with the said DNA under stringent conditions and has anability to transport carnitine and its analog in a sodium dependentmanner.

Still further, the invention relates to (4) an expression vector havingthe above-described gene or a gene encoding a protein of the said gene.

Also, the invention relates to (5) a host cell transformed by theabove-described expression vector.

Further, the invention relates to (6) a nucleotide including a partialsequence of consecutive 14 or more bases of the base sequencerepresented by SEQ ID NO. 1 or a complementary sequence thereof.

Still further, the invention relates to (7) a probe comprising theabove-described nucleotide and detecting the gene encoding a proteinhaving an ability to transport carnitine and its analog in a sodiumdependent manner.

Also, the invention relates to (8) a method of modulating an expressionof the gene encoding a protein having an ability to transport carnitineand its analog in a sodium dependent manner by using the above-describednucleotide.

Further, the invention relates to (9) an antibody to the protein definedin (1).

Still further, the invention relates to (10) a method of detecting afunction of subjected materials as a substrate to the ability of theprotein of transporting carnitine and its analog in a sodium dependentmanner, by using the protein defined in (1).

Also, the invention relates to (11) a method of adjusting kinetics ofcarnitine and its analog transported by the protein defined in (1) inthe testis and the epididymis, by modulating the ability of the saidprotein of transporting carnitine and its analog, by using the protein,its specific antibody, its promoter, or its inhibitor.

Further, the invention relates to (12) a method of adjusting aninfluence of carnitine and its analog transported by the protein definedin (1) on spermatids, by modulating the ability of the said protein oftransporting carnitine and its analog, by using the said protein, itsspecific antibody, its promoter, or its inhibitor.

Still further, the invention relates to (13) a method of adjusting aninfluence of carnitine and its analog transported by the protein definedin (1) on a fertilizability of spermatids, by modulating the ability ofthe said protein of transporting carnitine and its analog, by using theprotein, its specific antibody, its promoter, or its inhibitor.

Also, the invention relates to (14) a method of detecting and adjustingan influence of carnitine and its analog transported by the proteindefined in (1) on a fertilizability of spermatids or fertilizability ofan individual, by making the protein express in an excessive amount in aspecific cell or modulating the ability of the said protein existing inthe cell of transporting carnitine and its analog, by using the saidprotein, its specific antibody, its promoter, or its inhibitor.

Namely, the inventors have isolated the four organic anion transportersOAT1, OAT2, OAT3, and OAT4 as described above. Amino acid sequences ofthese organic anion transporters are about 40% homologous with eachother. The inventors have investigated EST (expressed sequence tag)database based on these sequences to identify a novel cDNA fragmentwhich is homologous with OAT1, OAT2, OAT3and OAT4. Using the cDNAfragment, the inventors have identified a novel clone (CT2) which hadnot been reported according to the human testis cDNA library toaccomplish the present invention.

The carnitine transporter CT2 of the present invention comprises anability of transporting L-carnitine and its analog from one cell toanother through a cell membrane, and a narrow substrate selectivitycomparing other drug transporters belonging to the OAT family or the OCTfamily.

The protein of the present invention includes a protein having the aminoacid sequence represented by SEQ ID NO: 2 and a protein having the aminoacid sequence derived from the said amino acid sequence by deletion,substitution, or addition of one to several amino acids. An amount ofthe deletion, the substitution, or the addition of the amino acid(s) maybe to such an extent that organic anion transport activity is not lost,and the number of the amino acids may generally be from 1 to about 110,preferably from 1 to about 55. Amino acid sequence of each of theseproteins has a homology to the amino acid sequence represented by SEQ IDNO: 2 of generally 75%, preferably 90%.

The gene of the present invention includes a gene comprising a DNAhaving a base sequence represented by SEQ ID NO. 1 and a gene comprisinga DNA encoding a protein which hybridizes with the DNA having the basesequence represented by SEQ ID NO. 1 under stringent conditions and hasan ability to transport carnitine and its analog in a sodium dependentmanner.

The hybridization under stringent conditions of the present inventionmay be conducted in a hybridization solution of 5×SSC (standard salinecitrate) or a hybridization solution having a salt concentrationequivalent to 5×SSC under a temperature condition of from 37° C. to 42°C. for about 12 hours and then preliminarily washed with a solution of5×SSC, or a solution having a salt concentration equivalent to 5×SSC,followed by washing with a solution of 1×SSC or a solution having a saltconcentration equivalent to 1×SSC. The higher stringency can be achievedby conducting the washing with a solution of 0.1×SSC or a solutionhaving a salt concentration equivalent to 0.1×SSC.

The carnitine transporter gene and the protein of the present inventioncan be isolated and obtained by screening using an organ, a tissue, or acultured cell of a suitable mammal as a gene source. The mammal includesnon-human animals; such as a dog, a cow, a horse, a goat, a sheep, amonkey, a pig, a rabbit, a rat, and a mouse; and a human.

The screening and isolation of the gene may preferably be conducted by ahomology screening, a PCR screening or the like.

The base sequence of the obtained cDNA may be determined by an ordinarymethod to analyze a translation region, whereby determining an aminoacid sequence of a protein, CT2, encoded by the base sequence.

It is possible to verify that the obtained cDNA is the cDNA of acarnitine transporter, namely, the gene product encoded in the cDNA is acarnitine transporter, by the following manner, for example.

It is possible to confirm the ability to transport (uptake) carnitineinto a cell by expressing a cRNA prepared from the obtained CT2cDNA byintroducing the cRNA into an oocyte and measuring a substrate uptakeinto the cell employing an ordinary uptake experiment (Sekine, T. etal., Biochem. Biophis. Res. Commun., Vol. 251, pp. 586-591, 1998)wherein carnitine is used as a substrate.

Also, it is possible to investigate a transport characteristic and asubstrate specificity of the CT2 by applying a similar uptake experimentto the expressed cell.

Further, it is possible to investigate characteristics of the CT2 suchas a time dependent transport, a sodium dependence, a substrateselectivity, and a pH dependence by applying a similar uptake experimentto the expressed cell.

By screening a suitable cDNA library or a genomic DNA library preparedfrom a different gene source using the obtained cDNA of the CT2 gene, itis possible to isolate a homologous gene, a chromogene, or the like,derived from a different tissue or a different organism.

Also, by using a synthetic primer designed based on disclosedinformation of the base sequence of the gene of the present invention(represented by SEQ ID NO. 1 or a part thereof), it is possible toisolate the gene from the cDNA library according to an ordinary PCRmethod.

DNA libraries such as the cDNA library and the genomic DNA library canbe prepared by the method. For example, described in Molecular Cloning;written by Sambrook, J, Fritsh, E. F. and Maniatis, T.; published byCold Spring Harbor Laboratory Press in 1989. Alternatively, acommercially available library may be used when it is purchasable.

In order to obtain a human genomic structure of the CT2 gene, thegenomic DNA library is screened using the cDNA of the obtained CT2 geneto analyze the clone obtained by the screening. Alternatively, it may besearched using a homology search program on the basis of publiclydisclosed information of human genome analysis results.

The carnitine transporter (CT2) of the present invention can be producedby a gene recombinant technology using, for example, the cDNA encodingthe carnitine transporter. For instance, the DNA (such as cDNA) encodingthe carnitine transporter is incorporated into a suitable expressionvector, such as a plasmid vector, a phage vector, and a virus vector tointroduce the obtained recombinant DNA into a suitable host cell. Anexpression system (host vector system) for producing a polypeptideincludes expression systems of bacteria, yeast, an insect cell, and amammal cell. Among the above expression systems, it is desirable to usethe insect cell and the mammal cell for the purpose of obtaining afunctional protein.

For example, when expressing the polypeptide in the mammal, the DNAencoding the carnitine transporter is inserted downstream of a suitablepromoter (such as SV40 promoter, LTR promoter, and elongation 1 αpromoter) in a suitable expression vector (such as a plasmid vector, aretrovirus-based vector, a papillomavirus vector, a vacciniavirusvector, and a SV40-based vector) to construct the expression vector.Then, a suitable animal cell is transformed by the obtained expressionvector, and the transformant is cultured in a proper medium to producethe desired polypeptide. The host mammal cells includes a monkey COS-7cell, a Chinese hamster CHO cell, a human Hela cell or a cell strain ofa primary culture cell derived from the kidney tissue, a pigkidney-derived LLC-PK1 cell, an opossum kidney-derived OK cell,mouse-derived proximal convoluted tubules S1, S2, and S3 cells, and thelike.

Examples of the cDNA encoding the carnitine transporter CT2 includes,but not limited to, the cDNA having the base sequence represented by SEQID NO. 1, and, also, a DNA corresponding to the amino acid sequence andencoding the polypeptide may be used as the cDNA. In this case, it isknown that one amino acid is encoded by 1 to 6 kinds of codons, and thecodon(s) to be used may be selected arbitrarily, but, it is desirable todesign a sequence which enables a higher level of expression in view offrequency of using codon of the host used for the expression. The DNAhaving the designed base sequence is obtainable by a DNA chemicalsynthesis, a combination with a fragment of the above-described cDNA, apartial alteration of the base sequence, or the like. Artificial partialalteration and mutation introduction of the base sequence can be carriedout by the site specific mutagenesis disclosed in Mark, D. F. et al.,Proc. Natl. Acad. Sci. USA, Vol. 18, pp. 5662-5666, 1984, using a primerconstituted of synthetic oligonucleotides encoding the desiredalteration.

The nucleotide (oligonucleotide or polynucleotide) which hybridizes withthe carnitine transporter gene of the present invention under stringentconditions can be used as the probe for detecting the carnitinetransporter gene and also as an antisense oligonucleotide, a ribozyme,or a decoy for modulating the carnitine transporter expression. Thenucleotide includes a nucleotide comprising a partial sequence ofconsecutive 14 or more bases of the base sequence represented by SEQ IDNO. 1 or a complementary sequence thereof, and, the longer sequence suchas that including 20 or more bases and 30 or more bases may be used asthe partial sequence in order to enhance specificity of thehybridization.

Further, by using the carnitine transporter of the present invention orits immunologically equivalent polypeptide, it is possible to obtain anantibody thereof, and the antibody can be used for carnitine transporterdetection and purification. The antibody can be produced by using thecarnitine transporter of the present invention, a synthetic peptidehaving a fragment or a partial sequence of the carnitine transporter, asan antigen. A polyclonal antibody can be produced by an ordinary methodof inoculating the antibody to a host animal (such as a rat and arabbit) and recovering an antiserum, and a monoclonal antibody can beproduced by an ordinary hybridoma method or the like.

By using the protein of the present invention, it is possible to detectfunction of subjected materials as a substrate to the ability of thesaid protein transporting carnitine and its analog in a sodium dependentmanner.

Further, by modulating the ability of the protein of the presentinvention of transporting carnitine and its analog using the protein,its specific antibody, its promoter (for example hormones such as anandrogen), or its inhibitor (such as an antisense nucleotide), it ispossible to adjust kinetics of carnitine and its analog transported bythe said protein in the testis and the epididymis.

Furthermore, by modulating the ability of the protein of the presentinvention of transporting carnitine and its analog using the protein,its specific antibody, its promoter (for example hormones such as anandrogen), or its inhibitor (such as an antisense nucleotide), it ispossible to adjust an influence on fertilizability of the spermatids ofcarnitine and its analog transported by the said protein.

Still further, by modulating the ability of the protein of the presentinvention of transporting carnitine and its analog using the protein,its specific antibody, its promoter (for example hormones such as anandrogen), or its inhibitor (such as an antisense nucleotide), it ispossible to adjust an influences on fertilizability of the spermatids oran individual of carnitine and its analog transported by the saidprotein.

In addition, by making the protein of the present invention express inan excessive amount in a particular cell, or by modulating the abilityof the said protein already existing in a cell of transporting carnitineand its analog using the protein, its specific antibody, its promoter(for example hormones such as an androgen), or its inhibitor (such as anantisense nucleotide), it is possible to detect or adjust an influenceon the fertilizability of spermatids or an individual by carnitine andits analog transported by the said protein.

EXAMPLES

The present invention will hereinafter be described in more detail byexamples, but it is not limited to these examples at all.

Note that operations in the following examples are performed inaccordance with the method described in Molecular Cloning: written bySambrook, J., Fritsh, E. F., and Maniatis, T.; published by Cold SpringHarbor Laboratory Press in 1989 or, when using a commercially availablekit, in accordance with instructions thereof unless otherwise noted.

Example 1

Isolation and Analysis of Carnitine Transporter (CT2) cDNA Specific toTestis and Epididymis

Based on base sequence information of OAT1, OAT2, OAT3, OAT4, and CT1,which were isolated by some of the inventors, publicly disclosed ESTdatabase was searched. As a result of this search, a novel cDNA fragmentAA778598 which is homologous with OAT1, OAT2, OAT3, OAT4, and CT1 wasobtained.

By using a probe labeled AA778598 with ³²P, a human testis cDNA libraryprepared in advance was screened. Hybridization was conducted in ahybridization solution at 50° C. all day and night, and then a filterfilm was washed with 0.1×SSC (standard saline citrate)/0.1% SDS (sodiumdodecyl sulfate) at 50° C. Used as the hybridization solution was abuffer solution of pH 6.5 containing 50% formamide, 5×SSC, 3×Denhardsolution, 0.2% SDS, 10% dextran sulfate, 0.2 mg/ml denatured sermonsperm DNA, 2.5 mM sodium pyrophosphate, 25 mM MES, and 0.01% Antifoam B(foam inhibitor; a product of Sigma-Aldrich, Co.). A clone isolated inλZipLox was subcloned to a plasmid vector pZL1 by an in vitro excisionmethod. As a result, a novel cDNA (CT2cDNA) having carnitine transportactivity was obtained.

Determination of a base sequence of the cDNA (CT2cDNA) obtained by theabove operation was performed by an automatic sequencer (product ofApplied Biosystems) using a specific primer (see SEQ ID NO.1).

Next, a cRNA (RNA complementary to cDNA) was prepared in vitro from aplasmid containing the CT2cDNA by using a T7RNA polymerase (see Sekine,T., et al., J. Biol. Chem., Vol. 272, pp. 18526-18529, 1997).

The thus-obtained cRNA was injected into oocytes of a platanna inaccordance with the already reported method (Sekine, T., et al., J.Biol. Chem., Vol. 272, pp. 18526-18529, 1997), and an uptake experimentby radioactive labeled L-carnitine was conducted on the oocytes. As aresult, it was proved that the oocytes which expressed the CT2 exhibit³H-L-carnitine uptake as shown in FIG. 1A. In contrast, uptake of¹⁴C-PAH (paraamino hippuric acid) which is a representative organicanion and uptake of ¹⁴C-TEA (tetraethyl ammonium) which is an organiccation were not observed.

Hereinafter, results of a transport function analysis, a study onsubstrate selectivity, an analysis of gene expression, an immunologicalstudy on the protein encoded by the gene in the testis and theepididymis, and a structure analysis of the CT2 gene in a human genome,all of which were conducted using the CT2 of the present invention, willbe described in order.

(1) An experiment on time dependence of the carnitine transport by CT2was conducted.

As a result of the experiment, the oocytes which expressed the CT2exhibited time dependence of ³H-L-carnitine uptake as shown in FIG. 1B.From this result, it was proved that the CT2 not only combines withcarnitine but also serves as a transporter transporting carnitine incells.

(2) A Michaelis-Menten kinetics experiment on the carnitine transport byCT2 was conducted.

A concentration dependence of the carnitine transport by CT2 was studiedthrough an investigation of changes in amounts of uptake of variousconcentration carnitines by CT2. The uptake experiment of radioactivelabeled carnitines was performed by using oocytes into which the CT2cRNAwas injected and in accordance with the above-described method. As aresult (FIG. 2A), a Km value of the L-carnitine uptake was about 20.3μM.

(3) An influence to be exerted by D-carnitine, which is an isomer ofL-carnitine, on the L-carnitine transport by CT2 was studied.

As shown in FIG. 2B, D-carnitine inhibited the L-carnitine transport byCT2 in a concentration dependent manner. A Ki value was about 30.1 μM.

(4) A sodium dependence of the CT2 in the carnitine transport wasstudied.

When extracellular sodium is substituted with lithium andN-methyl-D-glucamine, the carnitine transport by CT2 was decreased toreveal that the CT2 is an extracellular sodium dependent carnitinetransporter (FIG. 3A). However, the uptake was not perfectly decreasedwhen the extracellular sodium was substituted with lithium andN-methyl-D-glucamine, thereby it was revealed that CT2 is dependent on apartial extracellular sodium.

(5) A pH dependence of the CT2 in the carnitine transport was studied.

As shown in FIG. 3B, when an extracellular pH was inclined to acidity,the carnitine transport by CT2 was decreased.

(6) In order to study further on the substrate selectivity of CT2, inthe ³H-carnitine uptake experiment system by oocytes into which CT2cRNAwas injected, various carnitine analogs were added to the system and itsinfluences were investigated (inhibition experiment).

The ³H-carnitine uptake experiment was performed in accordance with theabove-described method using oocytes into which CT2cRNA was injected. 50nM ³H-carnitine uptake was measured in the presence or absence of 5 μMor 50 μM of various compounds (unlabelled). As a result, variouscarnitine analogs (D-carnitine, acetyl L-carnitine, acetyl DL-carnitine,octanoyl L-carnitine, betaine and the like) significantly inhibited the³H-carnitine transport by CT2 (FIG. 4). On the other hand, anionicsubstances and cationic substances such as paraamino hippuric acid andtetraethylammnonium did not show inhibition effect (FIG. 4). From theabove results, it was revealed that the CT2 is a specific transporter ofcarnitine and carnitine analogs.

(7) Expressions of CT2 gene in human tissues (northern blotting) wereanalyzed.

The full length of the CT2cDNA was labeled with ³²P-dCTP, and using itas a probe, hybridization of a filter (product of Clontech) blotted RNAsextracted from various human tissues was conducted. The hybridizationwas conducted overnight in a hybridization solution in which the fulllength of the CT2cDNA was contained, and then the filter was washed with0.1×SSC containing 0.1% SDS at 65° C. As a result of the northernblotting (FIG. 5), a strong band was detected only in the testis.

(8) An immunological study on a protein encoded by the CT2 gene in thetestis and the epididymis was conducted.

A polypeptide comprising 14 amino acid residues of a carboxyl terminalof the CT2 protein was synthesized, and it was immunized a rabbit toobtain an antiserum. A paraffin section of the human testis wassubjected to a deparaffinization and then treated with 3% hydrogenperoxide solution to inactivate endogenous peroxidase. The section wasincubated with an affinity purified polyclonal anti-CT2 antibody(primary antibody) for 24 hours, and then reacted with anti-rabbit IgGantibody labeled with peroxidase. Finally, by adding diaminobenzidine,which is a substrate for peroxidase, sites in which brown precipitationwas obtained were observed with an optical microscope. As shown in FIG.6, significant stains were observed in a cytoplasm of a Sertoli cell ofthe testis and a luminal side of an epithelial cell of the epididymis(an arrow in FIG. 6 indicates the CT2 expression site). Accordingly, theCT2 is a transporter which provides carnitine in Sertoli cells of thetestis and transports carnitine from blood to the lumens of theepididymis.

(9) A structure of the CT2 gene in a human genome was analyzed.

Publicly disclosed information of human genome analysis results wassearched by using a homology search program to find an exon-intronstructure of the CT2 gene. As shown in FIG. 7, the CT2 gene isconstituted of 10 exons, and a starting codon exists in the second exon.

INDUSTRIAL APPLICABILITY

The carnitine transporter specific to the testis and the epididymisselectively transporting carnitine and a gene thereof of the presentinvention enable an in vitro study on transport of carnitine and itsanalog at an expression site of the said transporter as well as aprediction of kinetics of the compounds in the genital organ based onthe study. Carnitine is the essential factor for producing energyrequired for mobility and fertilizability of the sperms, and it isconsidered that the invention of the said transporter will contribute toclarification of the cause of the idiopathic male infertility in future.Further, by clarifying a control factor modulating the expression of thesaid transporter, the invention can be applied to developments of amethod of modulating the ability of transporting carnitine and a methodof modulating the sperm fertilizability.

1. A method for screening a compound which inhibits or enhances thecarnitine transporting activity of the protein consisting of SEQ ID NO:2, the method comprising a) providing a cell having the functionalprotein consisting of SEQ ID NO: 2, b) contacting the cell withcarnitine, c) incubating the cell with the compound to be screened andd) detecting the carnitine transporting activity of the proteinconsisting of SEQ ID NO: 2 of the cell, wherein an inhibition orenhancement in the carnitine transporting activity of the proteinidentifies the compound being screened.